1. Field of the Invention
The present invention relates to nanoscale energy transfer, the conversion of chemical energy into light and, more particularly, to semiconductive quantum rods that posses rod-in-rod morphology that are conjugated with firefly luciferase.
2. Description of the Related Art
Research at the nanoscale biotic-abiotic interface centers on endowing an inorganic nanocrystal with the physical, chemical, or energetic properties of biosystems. This has led to an influx of biomimetic self-assembly, recognition, and energy transfer designs. One frontier that combines these advances is the ability to harness the energy converting properties of enzymes, by effectively transferring chemical energy from a substrate to an inorganic nanomaterial. This process could lead to revolutionary ways of converting chemical energy to light, for example, providing new lighting strategies, in-vivo signaling routes, and improved understanding of biosystems.
Quantum dots and rods have been proven to be powerful energy donors in a number of fluorescence resonance energy transfer (FRET) designs. This is due in large part to a broad absorption profile, size tunable emission, photostability, and long excited state lifetimes. However these same qualities limit the use of QDs as energy acceptors.
Bioluminescence energy transfer (BRET) on the other hand utilizes a bioluminescent enzyme in the presence of substrate to generate an excited state donor, and a fluorescent protein, dye, or QD 11-15 as the acceptor. For example, bioluminescent enzymes mutated from R. reniformis luciferase (Luc8) can be conjugated to semiconductive quantum dots (QDs) via EDC coupling. In the presence of the substrate colenterazine, the QD accepts the excited state energy of Luc8 via the non-radiative pathway known as bioluminescence resonance energy transfer (BRET). This allows for the blue-green emission of the enzyme to be converted to the red, and even near infrared (NIR) colors of the QD. BRET nanosystems have been used for in-vivo NIR imaging, where it has proven to be particularly useful due to low background signals and the lack of a direct excitation requirement. The metric for BRET efficiency is the BRET ratio (BR), where the emission of the acceptor and donor are compared. To date, typical BR using QD acceptors is limited to only 0.5˜2.0. This efficiency is much lower than comparable studies using fluorescent protein (BR=1˜4), or molecular fluorophore acceptors (BR=10˜14). The origins of the comparatively low BR with QDs is unknown, but may likely be due to relatively large spatial distances (r), as well as limited stoichiometry (n).
Due to the microsecond lifetimes of bioluminescent processes and the chemical origin of excitation, the QD in particular are ideal acceptors due to shorter lifetimes and broad absorption in the visible region. To date, QD based BRET nanosystems use blue-emitting R. reniformis luciferase (rLuc) expressed with multiple copies of surface exposed lysine residues, which were conjugated to free carboxylates at polymer wrapped QDs. BRET was observed for a number of QD colors, including near infrared emitters, which have proven to be particularly useful for in-vivo imaging. There remains an important need to improve BRET efficiency by tailoring of QD morphology and structure to better optimize and understand the BRET nanosystems.